In HIV infection a large frequency of HIV specific CD8+ and CD4+ T lymphocytes are dysfunctional and coincidentally show an increased expression of the negative regulator of T cell activation PD-1, PD1 ligation by PD-L1 and PD-L2 leads to the inhibition of most T cell functions including proliferation, cytokine production and survival. However, the molecular mechanisms downstream of PD-1 remain unknown. The major objective of this project is to define the molecular mechanisms that render T cells dysfunctional following PD-1 ligation and to identify strategies that will allow the rescue of such dysfunction in HIV specific CD8 cells. We will test the hypothesis that PD1 interferes with upstream TCR signaling pathways in order to affect all T cell functions. We have shown that PD-1 ligation leads to the phosphorylation of SHP-1 and SHP-2 phosphatases. Moreover PD1 crosslinking leads to the inhibition of p56Lck activation through the phosphorylation of the tyrosine Y505 by the inhibitory kinase Csk. In Specific Aim 1 we will determine the proximal signaling defect in PD-1 hi exhausted cells from HIV infected patients and assess whether Csk is recruited to the immunological synapse by PD1 ligation or whether it is released from Cbp/PAG by the SHP- 2 phosphatase. We will determine by quantitative immunoprecipitation if the inhibitory kinase Csk and the in phosphatases SHP-1 and SHP-2 are recruited to the TCR complex. Fluorescence (Forster) resonance energy transfer (FRET) experiments will be carried out to identify the partners involved in the negative regulation of p56Lck activity. The recruitment of these negative regulators will be assessed in cells showing varying degrees of dysfunction . We will use siRNAs specific for Csk, SHP-1, SHP-2 and Fyn-T and activated forms of Lck to rescue T cell function in T cells at the extremes of this gradient of dysfunction. In Aim 2, live cell imaging using total internal reflection fluorescence microscopy (TIRFM) will be used to visualize, (in collaboration with Core C) the dynamics of the immunological synapse formation upon PD-1 crosslinking in HIV specific CD8+ T cells showing varying degrees of dysfunction. . We will assess whether inhibition of the kinase Csk and the phosphatases SHP-112 will restore the integrity of the synapse as observed in efficiently activated T cells. Results emanating from this project may lead to the identification of novel therapeutic targets that can allow immune reconstitution during HIV infection.